Compositions and methods of enhancing immune responses to Eimeria or limiting Eimeria infection

ABSTRACT

Vaccine vectors and methods of using the vaccine vectors to enhance the immune response to an Apicomplexan parasite and reduce the morbidity or morality associated with subsequent infection are provided herein. The vaccine vectors include a polynucleotide encoding a Rhomboid polypeptide and optionally include an immune-stimulatory polypeptide suitably expressed on the surface of the vaccine vector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a national stage filing under 35 U.S.C. 371of International Application No. PCT/US2014/016359, filed Feb. 14, 2014,which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/764,681, filed Feb. 14, 2011, both of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted Sequence Listing in .txt format. The .txtfile contains a sequence listing entitled “2014-02-135658-00201_ST25.txt” created on Feb. 13, 2014 and is 40.3 kilobytes insize. The Sequence Listing contained in this .txt file is part of thespecification and is hereby incorporated by reference herein in itsentirety.

INTRODUCTION

Coccidiosis, an infectious disease of poultry, swine, and cattle causedby apicomplexan protozoan parasites (Eimeria spp. and related parasites)presents problems worldwide. Coccidiosis is among the top ten infectiousdiseases of poultry in terms of its economic impact on the poultryindustry with production losses estimated to be up to $2 billionannually. Other apicomplexan parasites also cause disease, includingPlasmodium, Cryptosporidium and Toxoplasma, which are the causationagents of malaria, cryptosporidiosis and toxoplasmosis, respectively.

Typical signs of coccidiosis include rapid loss of appetite, reductionin weight, diarrhea and acute mortality. Outbreaks in a flock occur uponexposure to high levels of pathogen and in most cases, coccidiosispredisposes birds to secondary bacterial infections. Traditional methodsof disease control include the administration of antibiotics andchemotherapeutic agents. However, with continuous usage, this has led toresistance issues. Antibiotic use also decreases social acceptance ofpoultry meat. Vaccination is a rational approach because of its abilityto confer long-term protection, typically for the entire lifespan ofcommercial chickens.

Most commercially available vaccines against Eimeria are based oncontrolled low dosage of essentially fully virulent but drug-sensitiveEimeria parasites. Vaccination with current Eimeria-based vaccinesproduces substantial vaccine-reaction morbidity and economic losses invaccinated flocks. Thus an effective low-virulence vaccine againstEimeria is needed. An effective vaccine for Eimeria based on conservedimmunogenic targets may also prove useful as a vaccine against otherapicomplexan parasites.

SUMMARY

A vaccine vector comprising a first polynucleotide sequence encoding anApicomplexan Rhomboid polypeptide and methods of using the same areprovided herein.

In one aspect, a vaccine vector comprising a first polynucleotideencoding an Apicomlexan Rhomboid polypeptide or an immunogenic fragmentthereof and a second polypeptide sequence encoding an immunostimulatorypolypeptide is disclosed. The Apicomplexan Rhomboid polypeptide and theimmunostimulatory polypeptide are suitably expressed on the surface ofthe vaccine vector. The Apicomplexan Rhomboid polypeptide may compriseSEQ ID NO: 1, SEQ 10 NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 37,SEQ ID NO: 38, an immunogenic fragment of at least one of SEQ ID NO:1-4, 37-38 or combinations of SEQ ID NO: 1-4 and 37-38. Theimmunostimulatory polypeptide may be a CD154 polypeptide capable ofbinding CD40 or an HMGB1 polypeptide. The CD154 polypeptides includefewer than 50 amino acids and comprise amino acids 140-149 of CD154 or ahomolog thereof.

In another aspect, a vaccine vector comprising a first polynucleotideencoding an Apicomlexan Rhomboid polypeptide of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 37, SEQ ID NO: 38, animmunogenic fragment of at least one at SEQ ID NO: 1-4 or 37-38 orcombinations of SEQ ID NO: 1-4 or 37-38. The Apicomplexan Rhomboidpolypeptide may be expressed on the surface of the vaccine vector.

Vaccine vectors according to the present invention may be a virus,yeast, bacterium, or liposome vector. Pharmaceutical compositions may becomprised of the vaccine vectors described herein and a pharmaceuticallyacceptable carrier.

In still another aspect, methods of enhancing the immune responseagainst an Apicomplexan parasite in a subject by administering a vaccinevector described herein to the subject are provided. The enhanced immuneresponse may be an enhanced antibody response, an enhanced T cellresponse or a combination thereof.

In a still further aspect, methods of reducing morbidity and mortalityassociated with infection with an apicomplexan parasite in a subject byadministering a vaccine vector as described herein to the subject areprovided. The vaccine vector is capable of reducing the morbidity andmortality associated with subsequent infection with an apicomplexanparasite in subjects administered the vaccine vector as compared tocontrols.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation showing the homology of the MPPsequence among several Apicomplexan parasites. The consensus MPPsequence is highly similar in amino acid sequences in the Apicomplexans.Positions that are not identical are indicated with an X in theconsensus sequence which is shown on the top line of the figure and isSEQ ID NO: 38. The Toxoplasma gondii sequences (the first four linesbelow the consensus) share 100% identity to the MPP sequence of SEQ IDNO: 2 from Eimeria maxima. The bottom two sequences are the homolog fromNeospora caninum (SEQ ID NO: 3) and Eimeria tenella (SEQ ID NO: 4),respectively.

FIG. 2 is a schematic representation of the vaccine vector constructsdescribed in the Examples.

FIG. 3 is a bar graph showing the body weight (grams) of the chickenseight days post-infection with Eimeria maxima after inoculation with theindicated vaccine vector expressing the indicated sequences. Significantdifferences (p<0.05) between treatment groups are indicated by differentletters.

FIG. 4 is a bar graph showing the body weight (grams) of the survivingchickens 29 days post-challenge infection with Eimeria maxima afterinoculation with the indicated vaccine vector expressing the indicatedsequences. Significant differences (p<0.05) between treatment groups areindicated by actual p values and an asterisk (*).

FIG. 5 is a bar graph showing the percent mortality in the face of avirulent challenge infection with Eimeria maxima at eight dayspost-challenge infection with Eimeria maxima after inoculation with theindicated vaccine vector expressing the indicated sequences. Significantdifferences (p<0.05) are indicated with an asterisk (*).

DETAILED DESCRIPTION

Conventional vaccines against coccidiosis are generally based onlive/attenuated parasites that are delivered in controlled numbers.However, the risk of infection is not eliminated because the parasitesare viable and capable of causing disease. Additionally, productioncosts for these types of vaccine are extremely high because it involvespassing the parasites through live birds, collecting them at regularintervals and ensuring an uninterrupted cold transit chain fromproduction to use at the hatchery or on the farm. With vaccination beinga critical control method, the use of recombinant vaccines may improvethe overall efficacy of coccidiosis-based vaccines while decreasing theproduction costs.

Species of Eimeria are highly immunogenic and are capable of stimulatingrobust host immune responses. The wide repertoire of antigens that arepart of this eukaryote are highly specialized in function and aresuitable targets for recombinant vaccine development. Sporozoites andmerozoites are the most motile stages of the parasite and areresponsible for initiating and sustaining an active infection. Invasionof these stages into intestinal epithelial cells is an essential processfor the parasite to continue its life-cycle within host cells. A highlyspecialized set of organdies located at the anterior (apical) end of theparasite is involved in transporting the numerous proteins required forthe translocation of these motile stages from the intestinal lumen intothe epithelial layer. This apical complex consists of a variety ofsecretory organelles including a large number of micronemes thattransport a milieu of proteins to the surface of motile apicomplexanzoites in support of the essential function of motility.

Among several well-described microneme-associated proteins,thrombospondin-related adhesive protein (TRAP) has been used as asuccessful recombinant antigen in Salmonella recombinant andBacillus-vectored systems as a vaccine candidate. See U.S. PublicationNo 2011/0111015, which is incorporated herein by reference in itsentirety. Many microneme proteins have a similar mode of action in thatthey a released from the microneme complex at the anterior end of thesporozoite as they approach a host cell and act as a link the parasiteand whatever substrate they are upon. The microneme protein is thentranslocated across the surface of the parasite posteriorly, therebymoving the parasite closer to the host cell. This gliding form ofmotility is typical of all apicomplex parasites. When the micronemeprotein has been translocated to the posterior end of the parasite, itneeds to be cleaved and released from the surface of the parasite inorder to successfully complete the invasion process. This function isperformed by a family of proteases that are constitutively expressedwithin or on the parasite cell membrane. The cleavage process occursintracellularly and is an absolute requirement for propagating theinfection.

A novel approach to recombinant vaccine design involves targeting thisprotease and interfering with the cleavage/invasion process. The familyof proteases that are involved in the cleavage process are calledrhomboid proteases and are extremely well-described in Toxoplasmaspecies with homologues in Eimeria and other Apicomplexa. Rhomboidproteases (ROM4 and ROM5, MPP) are centrally implicated in the cleavageof microneme proteins and share good homology among differentapicomplexan parasites. Our hypothesis was based on the premise that ifwe are able to immunologically target the protease, antibody bindingwould interfere with the cleavage process and thereby impairsporozoite/merozoite mobility. For successful infection to occur,intracellular development of the parasite is essential and our approachmay curtail cell invasion thus, interfering with establishment of thelife-cycle. One advantage of targeting MPP is that the conserved natureof this protein across many apicomplexan species makes it a suitabletarget not only for Eimeria, but other Apicomplexa as well.

Predicted antigenic regions of MPP (ROM5) were aligned and checked forhomology among six different Apicomplexa (FIG. 1). The seven sequencescompared are as follows: Eimeria tenella ROM4 (JN558353), Toxoplasmagondii ME49 ROM5 (XP_002370238), Toxoplasma gondii ROM5 (AAT84606),Toxoplasma gondii ROM5 (AY587208), Toxoplasma gondii RH ROM5 (AM055942),Toxoplasma gondii (AY634626), and the MPP insert from Eimeria Maxima ofSEQ ID NO: 2. Suitable Apicomplexan parasites include, but are notlimited to: Eimeria species, including but not limited to Eimeriatenella, Eimeria maxima, and Eimeria brunetti; Toxoplasma gondii;Neospora caninum; Cryptosporidium species; and Plasmodium species,including but not limited to Plasmodium falciparum, Plasmodium malariae,Plasmodium knowlesi, and Plasmodium vivax.

Recombinant DNA technologies enable relatively easy manipulation of manyyeast, bacterial and viral species. Some microorganisms are mildlypathogenic or non-pathogenic, but are capable of generating a robustimmune response. These microorganisms make attractive vaccine vectorsfor eliciting an immune response to antigens recombinantly expressed inthe vector. Vaccines vectored by microorganisms may mimic a naturalinfection, help produce robust and long lasting mucosal immunity, andmay be relatively inexpensive to produce and administer. In addition,such vectors can often carry more than one antigen and have potential toprovide protection against multiple infectious agents.

In one aspect, a vaccine vector comprising a first polynucleotidesequence encoding an Apicomplexan Rhomboid polypeptide of SEQ ID NO:1-4, 37-38, an immunogenic fragment thereof or combinations thereof isprovided. In another embodiment, the vaccine vector may include a firstpolynucleotide encoding an Apicomplexan Rhomboid polypeptide and asecond polynucleotide encoding an immunostimulatory polypeptide isprovided. The Rhomboid polypeptide and the optional immunostimulatorypolypeptide are expressed on the surface of the vaccine vector. TheRhomboid polypeptide may comprise the full-length protein (SEQ ID NO:39) or an immunogenic fragment such as those provided in SEQ ID NO: 1-4and 37-38. For example, the Rhomboid polypeptide may comprise, mayconsist essentially of or may consist of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 37, SEQ ID NO: 38 or an immunogenicfragment of any of these SEQ ID NOs. Combinations of these fragments mayalso be used in a vaccine vector. A vaccine vector may include SEQ IDNO: 1-4 or 37-38. A single vaccine vector may include multiple copies ofa single fragment as well.

The immunogenic fragment of a Rhomboid polypeptide may be a sequencethat is at least 5, 6, 7, 8, 10, 12, 14, 16, 18 or 20 amino acids longand has at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% percentidentity to the fragments of SEQ ID NO: 1-4 or 37-38 provided herein.Without being limited by theory, the vaccine vectors provided herein arebelieved to be reducing morbidity and mortality associated with Eimeriainfection by inducing an antibody response that is capable of blockinginvasion of the parasites into cells. Those of skill in the art areaware that B cells epitopes are often hydrophilic in nature and thisinformation can be used to generate immunogenic fragments to thepolypeptides of SEQ ID NO: 1-4 and 37-38 provided herein. Ahydrophilicity plot of SEQ ID NO: 2 reveals three hydrophilic areas ofthe peptide and three potential B cell epitopes including amino acids1-11, 18-27 and 31-43 of SEQ ID NO: 2. These amino acid fragmentscorrespond to amino acids 7-16 of SEQ ID NO: 3 and 37 and amino acids12-21 of SEQ ID NO: 4. As shown by the two consensus sequences of SEQ IDNO: 1 and SEQ ID NO: 38, amino acids corresponding to 18-27 of SEQ IDNO: 2 are highly conserved across species and genera of Apicomplexanparasites. An immune response to such a highly conserved epitope mayallow for cross species or even cross genera immunity from a singlevaccine.

A vaccine includes any composition comprising a polynucleotide encodingan antigenic polypeptide that is capable of eliciting an immune responseto the polypeptide. A vaccine vector is a composition that can beengineered to carry antigens or immunostimulatory polypeptides and mayalso comprise an adjuvant or be administered with an adjuvant to furtherincrease the immune response to the parasite and provide betterprotection from morbidity and mortality associated with a subsequentinfection. The use of vectors, such as bacterial vectors, forvaccination and generation of immune responses against Eimeria or otherapicomplexan parasites such as Plasmodium (the causative agent ofmalaria), Toxoplasma and Cryptosporidium is disclosed. The immuneresponses after administration of the vaccine vector need not be fullyprotective, but may decrease the morbidity or percentage mortality (i.e.likelihood of mortality) associated with subsequent infection.

Polynucleotides encoding Rhomboid polypeptide antigens of SEQ ID NO:1-4, 37-38 or fragments thereof and other antigens from any number ofpathogenic organisms may be inserted into the vector and expressed inthe vector. The expression of these polynucleotides by the vector willallow generation of antigenic polypeptides following immunization of thesubject. The polynucleotides may be inserted into the chromosome of thevector or encoded on plasmids or other extrachromosomal DNA. Those ofskill in the art will appreciate that numerous methodologies exist forobtaining expression polynucleotides in vectors such as Salmonella orBacillus. The polynucleotides may be operably connected to a promoter(e.g., a constitutive promoter, an inducible promoter, etc.) by methodsknown to those of skill in the art. Suitably, polynucleotides encodingthe Rhomboid antigens are inserted into a vector, e.g., a bacterialvector, such that the polynucleotide is expressed.

The polynucleotides encoding the Rhomboid antigens may be inserted inframe in a polynucleotide encoding a transmembrane protein. Thepolynucleotide encoding the Rhomboid antigen is inserted into the vectorpolynucleotide sequence to allow expression of the Rhomboid antigen onthe surface of the vector. For example, the polynucleotide encodingRhomboid antigen may be inserted in frame into the vector polynucleotidein a region encoding an external loop region of a transmembrane proteinsuch that the vector polynucleotide sequence remains in frame. In oneembodiment, the first polynucleotide encoding the Rhomboid polypeptidemay be inserted into loop 9 of the lamB gene of Salmonella.

In another embodiment, the first polynucleotide is inserted into or at asurface exposed end of a protein that is attached to the cell wall, butis not a transmembrane protein. The protein may be a secreted proteinthat is anchored or attached to the cell wall via as protein or lipidanchor. In the Examples, the MPP (SEQ ID NO: 2) polypeptide is insertedat the 3′ end of the fibronectin binding protein (FbpB) of Bacillussubtilis. Alternatively, the first polynucleotide encoding the Rhomboidantigen may be inserted into a polynucleotide encoding a secretedpolypeptide.

Those of skill in the art will appreciate that the polynucleotideencoding the Rhomboid antigen could be inserted in a wide variety ofvector polynucleotides to provide expression and presentation of theRhomboid antigen to the immune cells of a subject treated with thevaccine. The polynucleotide encoding the Rhomboid antigen may beincluded in a single copy or more than one copy. The multiple copies maybe inserted in a single location or more than one location.Alternatively, multiple epitopes such as combinations of the Rhomboidantigens provided herein as SEQ ID NO: 1-4 and 37-38 or combinations ofthis epitope with other apicomplexan epitopes such as TRAP or epitopesfrom other pathogens may be inserted into the vector at the same or morethan one location.

Suitably the first polynucleotide encodes a portion of the Rhomboidpolypeptide, the entire Rhomboid polypeptide or more than one epitopefrom the Rhomboid polypeptide. The combination of epitopes from morethan one polypeptide from a single parasite or pathogen or thecombination of epitopes from related pathogens is specificallycontemplated. The polynucleotide may be inserted into the vector and maybe inserted as a fusion protein containing more than a single epitope.In the Examples, SEQ ID NOs: 2 and 15 (MPP-HMGB1) or SEQ ID NOs: 2, 40and 15 (MPP-TRAP-HMGB1) were incorporated into a Bacillus vector.Suitably, the portion of the Rhomboid polypeptide inserted into thevector is an antigenic fragment. An antigenic fragment is a peptide orpolypeptide capable of eliciting as cellular or humoral immune responseor capable of reducing the morbidity or mortality associated withsubsequent infection with the parasite.

An antigenic polypeptide or epitope includes any polypeptide that isimmunogenic. The antigenic polypeptides include, but are not limited to,antigens that are pathogen-related, allergen-related, tumor-related ordisease-related. Pathogens include viral, parasitic, fungal andbacterial pathogens as well as protein pathogens such as the prions. Theantigenic polypeptides may be full-length proteins or portions thereof.It is well established that immune system recognition of many proteinsis based on a relatively small number of amino acids, often referred toas the epitope. Epitopes may be only 4-8 amino acids long. Thus, theantigenic polypeptides described herein may be full-length proteins,four amino acid long epitopes or any portion between these extremes. Infact the antigenic polypeptide may include more than one epitope from asingle pathogen or protein. The antigenic polypeptides may have, atleast 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% percent identity tothe SEQ ID NOs provided herein. Suitably, an antigenic fragment of theRhomboid antigen or polypeptide may be four, five, six, seven, eight,nine, 10 or more amino acids, 15 or more amino acids or 20 car moreamino acids of the full-length protein sequence.

Multiple copies of the same epitope or multiple epitopes from the sameor different proteins may be included in the vaccine vector. Theepitopes in the vaccine vector may be related and homologous to allowtargeting of multiple related pathogens with a single vaccine vector. Itis envisioned that several epitopes or antigens from the same ordifferent pathogens or diseases may be administered in combination in asingle vaccine vector to generate an enhanced immune response againstmultiple antigens. Recombinant vaccine vectors may encode antigens frommultiple pathogenic microorganisms, viruses or tumor associatedantigens. Administration of vaccine vectors capable of expressingmultiple antigens has the advantage of inducing immunity against two ormore diseases at the same time, providing broader protection againstmultiple strains of a single pathogen or a more robust immune responseagainst as single pathogen.

In the examples, the MPP antigen (SEQ ID NO: 2) was co-expressed inseveral of the vectors with a second antigenic polypeptide. A highmolecular mass, asexual stage antigen from Eimeria maxima (EmTFP250) wasdemonstrated to be a target for maternal antibodies produced by breedinghens infected with this protozoan parasite. Analysis of the amino acidsequence of the antigen revealed a novel member of the TRAP(thrombospondin-related anonymous protein) family, containing 16thrombospondin type-1 repeats and 31 epidermal growth factor-likecalcium binding domains. See U.S. Patent Publication No. 2011/0111015.EmTFP250 or TRAP also contains two low complex, hydrophilic regions richin glutamic acid and glycine residues, and a transmembranedomain/cytosolic tail associated with parasite gliding motility that ishighly conserved within apicomplexan microneme proteins. Severalpotential epitopes were selected and are identified in SEQ ID NO: 1-3and 11 of U.S. Patent Publication No. 2011/0111015 which are reproducedherein as SEQ ID NO: 5-8, SEQ ID NO: 40 was used in the Examplesprovided herein and is referred to as a TRAP antigen as well. SEQ ID NO:40 and SEQ ID NO: 6 vary by a single amino acid. A proline at position 6of SEQ ID NO: 6 is changed to an arginine at the same position 6 of SEQID NO: 40. This change was made to make the epitope more flexible andhydrophilic with the goal of making it a better antigen. Those of skillin the art may make other single amino acids changes to improveantigenicity within the scope of this invention. Due to the conservednature of this antigen, expression of these epitopes by a vector mayinduce protective immunity against multiple apicomplexan parasites andadministration of a vaccine vector comprising two distinct antigenicpolypeptides may induce a more robust immune response.

Those of skill in the art will appreciate that the antigenicpolypeptides from other pathogens may be used in the vaccine vectors toenhance the immune response against more than one pathogen by a singlevaccine. It would be advantageous to administer a single vaccinedirected against multiple pathogens. A vaccine capable of eliciting animmune response to an Apicomplexan parasite, such as Eimeria, incombination with Influenza, Salmonella, Campylobacter or other pathogensis envisioned.

For example, the second antigenic polypeptide may be an Influenzapolypeptide, suitably it is an Influenza H5N1 polypeptide or apolypeptide associated with multiple strains of the Influenza virus suchas a polypeptide of the Influenza M2 protein. The ectodomain of theInfluenza A virus M2 protein, known as M2e, protrudes from the surfaceof the virus. The M2e portion of the M2 protein contains about 24 aminoacids. The M2e polypeptide varies little from one isolate to the nextwithin Influenza. In fact, only a few naturally occurring mutations inM2e have been isolated from infected humans since the 1918 flu epidemic.In addition, influenza viruses isolated from avian and swine hosts havedifferent, yet still conserved, M2e sequences. For reviews of the M2epolypeptide sequences isolated from human, avian and swine hosts see Liuet al., Microbes and Infection 7:171-177 (2005) and Reid et al., J.Viral. 76:10717-10723 (2002) each of which are incorporated herein byreference in its entirety. Suitably the entire M2e polypeptide may beinserted into the vaccine vector or only a portion may be used. An eightamino acid polypeptide (LM2 having amino acid sequence: EVETPIRN, SEQ IDNO: 9 or its variant M2eA having amino acid sequence EVETPTRN, SEQ IDNO: 10) was incorporated into a vaccine vector and demonstrated toproduce an antibody response after administration to chickens. See U.S.Publication No 2011/0027309 which is incorporated herein by reference inits entirety.

Other suitable epitopes for inclusion in an Influenza A vaccine vectorinclude, but are not limited to, polypeptides of the hemagglutinin (HA)or the nuclear protein (NP) of Influenza A. For example, the peptides ofSEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14 may beincluded in a vaccine vector. One of skill in the art will appreciatethat any of these sequences may be used in combination with any otherepitope including epitopes derived from other pathogens or antigens.

Immunostimulatory molecules included as part of the vaccine vector couldpotentially activate parts of the immune system critical to long-lastingprotection or provide an adjuvant effect. Immunostimulatorypolypepetides may be polypeptides capable of stimulating a naïve oradaptive immune response. The immunostimulatory polypeptides are notnatively associated with the vaccine vector and are polypeptidesnatively associated with a vertebrate immune system, such as that of thesubject to which the vaccine will be administered. Two immunostimulatorypolypeptides are described herein namely CD154 and High Mobility GroupBox 1 (HMGB1) polypeptides, but one of skill in the art will appreciatethat other immunostimulatory polypeptides could be used or alternativelycould be used in combination with those described herein.

Additional polynucleotides encoding polypeptides involved in triggeringthe immune system may also be included in a vaccine vector. Thepolynucleotides may encode immune system molecules known for theirstimulatory effects, such as an interleukin, Tumor Necrosis Factor,interferon complement, or another polynucleotide involved inimmune-regulation. The vaccine may also include polynucleotides encodingpeptides known to stimulate an immune response, such as the CD154 orHMGB1 polypeptides described herein.

HMGB1 is secreted by activated macrophages and damaged cells, and actsas a cytokine mediator of inflammation, affecting the innate immuneresponse. Portions of the HMGB1 sequence have been included in thevaccine vectors described in the Examples. The HMGB1 (High MobilityGroup Box-1) protein was first identified as a DNA-binding proteincritical for DNA structure and stability. It is a ubiquitously expressednuclear protein that binds DNA with no sequence specificity. The proteinis highly conserved and found in plants to mammals. The zebrafish,chicken and human HMGB1 amino acid sequences are provided in SEQ ID NO:23, SEQ ID NO: 15 and SEQ ID NO: 22, respectively. The sequencethroughout mammals is highly conserved with 98% amino acid identity andthe amino acid changes are conservative. Thus an HMGB1 protein from onespecies can likely substitute for that from another speciesfunctionally. The full-length HMGB1 protein or a portion thereof may beused as the HMGB1 polypeptide in the vaccine vectors described herein.HMGB1 has two DNA binding regions termed A box as shown in SEQ ID NO: 16and 17 and 13 box as shown in SEQ ID NO: 18 and 19. See Andersson andTracey, Annu. Rev. Immunol, 2011, 29:139-162, which is incorporatedherein by reference in its entirety.

HMGB1 is a mediator of inflammation and serves as a signal of nucleardamage, such as from necrotic cells. HMGB1 can also be actively secretedby cells of the monocyte/macrophage lineage in a process requiringacetylation of the protein, translocation across the nucleus andsecretion. Extracellular HMGB1 acts as a potent mediator of inflammationby signaling via the Receptor for Advanced Glycated End-products (RAGE)and via members of the Toll-like Receptor family (TLR), in particularTLR4. The RAGE binding activity has been identified and requires thepolypeptide of SEQ ID NO: 20. TLR4 binding requires the cysteine atposition 106 of SEQ ID NO: 15, which is found in the B box region ofHMGB1.

The inflammatory activities of HMGB1 do not require the full-lengthprotein and functional fragments have been identified. The B box hasbeen shown to be sufficient to mediate the pro-inflammatory effects ofHMGB1 and thus SEQ ID NO: 18 and 19 are HMGB1 polypeptides or functionalfragments thereof within the context of the present invention. Inaddition, the RAGE binding site and the pro-inflammatory cytokineactivity have been mapped to SEQ ID NO: 20 and SEQ ID NO: 21,respectively. Thus, these polypeptides are functional fragments of HMGB1polypeptides in the context of the present invention.

Those of skill in the art are capable of identifying HMGB1 polypeptidesand fragments thereof capable of stimulating pro-inflammatory cytokineactivity, using methods such as those in International Publication No.WO02/092004, which is incorporated herein by reference in its entirety.Suitably, the HMGB1 polypeptide includes the RAGE binding domain atamino acids 150-183 of SEQ ID NO:15 (SEQ ID NO: 20 or a homolog thereof)and the pro-inflammatory cytokine activity domain between amino acids89-109 of SEQ NO: 15 (SEQ ID NO: 21 or a homolog thereof). Inparticular, HMGB1 polypeptides and functional fragments or homologsthereof include polypeptides identical to, or at least 99% identical, atleast 98% identical, at least 97% identical, at least 96% identical, atleast 95% identical, at least 90% identical, at least 85% identical, orat least 80% identical to the HMGB1 polypeptides of SEQ ID NOs: 15 or16-23.

As described in more detail below, a vaccine vector may include a CD154polypeptide that is capable of binding CD40 in the subject andstimulating the subject to respond to the vector and its associatedantigen. Involvement of dendritic cells (DCs) is essential for theinitiation of a powerful immune response as they possess the uniqueability to activate naïve T cells, causing T cell expansion anddifferentiation into effector cells. It is the role of the DC, which isan antigen presenting cell (APC) found in virtually all tissues of thebody, to capture antigens, transport them to associated lymphoid tissue,and then present them to naïve T cells. Upon activation by DCs, T cellsexpand, differentiate into effector cells, leave the secondary immuneorgans, and enter peripheral tissues. Activated cytotoxic T cells (CTLs)are able to destroy virus-infected cells, tumor cells or even APCsinfected with intracellular parasites (e.g., Salmonella) and have beenshown to be critical in the protection against viral infection. CD40 isa member of the TNF-receptor family of molecules and is expressed on avariety of cell types, including professional antigen-presenting cells(APCs), such as DCs and B cells. Interaction of CD40 with its ligandCD154 is extremely important and stimulatory for both humoral andcellular immunity. Stimulation of DCs via CD40, expressed on the surfaceof DCs, can be simulated by anti-CD40 antibodies. In the body, however,this occurs by interaction with the natural ligand for CD40 (i.e. CD154)expressed on the surface of activated T-cells. Interestingly, theCD40-binding regions of CD154 have been identified. The CD40-bindingregion of CD154 may be expressed on the surface of a vector, such as aSalmonella or Bacillus vector, and results in an enhanced immuneresponse against a co-presented peptide sequence as shown in theExamples provided herein and in U.S. Patent Publication No.2011/0027309, which is incorporated herein by reference in its entirety.A CD154 polypeptide may be a portion of CD154 full-length protein or theentire CD154 protein. Suitably, the CD154 polypeptide is capable ofbinding CD40.

As discussed above, a CD154 polynucleotide encoding a CD154 polypeptidethat is capable of enhancing the immune response to the antigen may beincluded in the vaccine. Suitably, the CD154 polypeptide is fewer than50 amino acids long, more suitably fewer than 40, fewer than 30 or fewerthan 20 amino acids in length. The polypeptide may be between 10 and 15amino acids, between 10 and 20 amino acids or between 10 and 25 aminoacids in length. The CD154 sequence and CD40 binding region are nothighly conserved among the various species. The CD154 sequences ofchicken and human are provided in SEQ ID NO: 24 and SEQ ID NO: 25,respectively.

The CD40 binding regions of CD154 have been determined for a number ofspecies, including human, chicken, duck, mouse and cattle and are shownin SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQID NO: 30, respectively. Although there is variability in the sequencesin the CD40 binding region between species, the human CD154 polypeptidewas able to enhance the immune response in chickens. Therefore, one maypractice the invention using species specific CD154 polypeptides or aheterologous CD154 polypeptide. Thus the CD154 polypeptides or SEQ IDNO: 24-30 may be included in a vaccine vector or a polypeptide at least99, 98, 97, 96, 95, 93, 90 or 85% identical to the sequences of SEQ IDNO: 24-30 may be included in a vaccine vector.

The polypeptide from CD154 stimulates an immune response at least inpart by binding to its receptor, CD40. A polypeptide homologous to theCD154 polypeptide which is expressed on immune cells of the subject andwhich is capable of binding to the CD40 receptor on macrophages andother antigen presenting cells. Binding of this ligand-receptor complexstimulates macrophage (and macrophage lineage cells such as dendriticcells) to enhance phagocytosis and antigen presentation while increasingcytokine secretions known to activate other local immune cells (such asB-lymphocytes). As such, molecules associated with the CD154 peptide arepreferentially targeted for immune response and expanded antibodyproduction.

The antigenic polypeptides and the immunostimulatory polypeptides aredelivered via a vaccine vector. The vaccine vectors may be bacterial,yeast, viral or liposome-based vectors. Potential vaccine vectorsinclude, but are not limited to Bacillus (Bacillus subtilis), Salmonella(Salmonella enteritidis), Shigella, Escherichia (E. coli), Yersinia,Bordetella, Lactococcus, Lactobacillus, Streptococcus, Vibrio (Vibriocholerae), Listeria, yeast such as Saccharomyces, or Pichia, adenovirus,poxvirus, herpesvirus, alphavirus, and adeno-associated virus. Livebacterial, yeast or viral vaccine vectors still pose risks toimmunocompromised individuals and require additional regulatoryscrutiny. Thus use of vectors that are killed or inactivated or qualifyas Generally Recognized As Safe (GRAS) organisms by the Food and DrugAdministration (FDA) is desirable. The problem is generating a robustimmune response using such vectors. Methods of inactivating or killingbacterial, yeast or viral vaccine vectors are known to those of skill inthe art and include, but are not limited to methods such as formalininactivation, antibiotic-based inactivation, heat treatment and ethanoltreatment. By including an immunostimulatory polypeptide such as HMGB1(high mobility group box 1) polypeptide on the surface of the vaccinevector we can generate a robust immune response against an apicomplexanparasite using a Bacillus spp. vector. In fact, the Examples demonstratethat this vector can be inactivated such that it cannot replicate andstill elicit a robust immune response after administration. The vaccinevectors may be wild-type bacteria, yeasts or viruses that are notpathogenic. Alternatively the vectors may be attenuated such that thevector has limited ability to replicate in the host or is not capable ofgrowing without supplemented media for more than a few generations.Those of skill in the art will appreciate that there are a variety ofways to attenuate vectors and means of doing so.

At least a portion of the antigenic polypeptide and at least a portionof the immunostimulatory polypeptide are present or expressed on thesurface of the vaccine vector. Present on the surface of the vaccinevector includes polypeptides that are comprised within an external loopof as transmembrane protein, interacting with, e.g., covalently orchemically cross-linked to, a transmembrane protein, a membrane lipid ormembrane anchored carbohydrate or polypeptide. A polypeptide can becomprised within a transmembrane protein by having the amino acidscomprising the polypeptide linked via a peptide bond to the N-terminus,C-terminus or anywhere within the transmembrane protein (i.e. insertedbetween two amino acids of the transmembrane protein or in place of oneor more amino acids of the transmembrane protein (i.e.deletion-insertion). Suitably, the polypeptides may be inserted into anexternal loop of a transmembrane protein. Suitable transmembraneproteins are srtA, cotB and lamB, but those of skill in the art willappreciate many suitable transmembrane proteins are available.Polypeptides may be linked to a membrane or cell wall anchored proteinor lipid such that the antigenic polypeptide and the immunostimulatorypolypeptide are expressed on the surface of the vaccine vector.

As described above, polynucleotides encoding the antigenic orimmunostimulatory polypeptides may be inserted into the chromosome ofthe vector or maintained extrachromosomally (e.g., on a plasmid, BAC orYAC). Those of skill in the art will appreciate that thesepolynucleotides can be inserted in frame in a variety of polynucleotidesand expressed in different parts of the vector or may be secreted. Thepolynucleotide encoding the immunostimulatory polypeptide capable ofenhancing the immune response to the antigenic polypeptide may alsoencode the antigenic polypeptide. The polynucleotide encoding theantigenic polypeptide may be linked to the polynucleotide encoding theimmunostimulatory polypeptide, such that in the vector, the twopolypeptides are portions of the same polypeptide, such as in a fusionprotein. In the Examples, a polynucleotide encoding the antigenicpolypeptide also encodes the immunostimulatory polypeptide. In oneembodiment, the two polynucleotides encoding the polypeptides are bothinserted in frame in loop 9 of the lamB gene of Salmonella enteritidisor another vaccine vector. Those of skill in the art will appreciatethat bacterial polynucleotides encoding other transmembrane proteins andother loops of the lamB gene may also be used.

Alternatively, the polynucleotide encoding the antigenic polypeptideand/or the immunostimulatory polypeptide may be inserted into a secretedpolypeptide that is displayed or presented on the surface of the vaccinevector through association with a protein, lipid or carbohydrate on thesurface of the vaccine vector. Those of skill in the art will appreciatethat the polynucleotide encoding the antigenic polypeptide and/or theimmunostimulatory polypeptide could be inserted in a wide variety ofvaccine vector polynucleotides to provide expression and presentation ofthe antigenic polypeptide and or the immunostimulatory polypeptide tothe immune cells of a subject treated with the vaccine vector byexpression on the surface of the vaccine vector. The coding region ofthe Apicomplexan Rhomboid polypeptide and the immunostimulatorypolypeptide can be fused to the C-terminus of the Staphylococcus aureusfibronectin binding protein containing a sorting motif for sortase fromListeria. This allows the secreted proteins to be anchored on the cellwall of gram positive bacteria such as Bacillus. See Nguyen andSchumann, J Biotechnol (2006) 122: 473-482, which is incorporated hereinby reference in its entirety. This system was used in the Examples toallow expression of the Rhomboid polypeptide linked to HMGB1 on thesurface of Bacillus. Other similar methods may also be used.

Alternatively, the polypeptides may be covalently or chemically linkedto proteins, lipids or carbohydrates in the membrane, cell wall, orcapsid if a viral vector is being used through methods available topersons of skill in the art. For example, di-sulfide bonds orbiotin-avidin cross-linking could be used to present the antigenic andimmunostimulatory polypeptides on the surface of a vaccine vector.Suitably, the antigenic polypeptide and the immunostimulatorypolypeptide are part of a fusion protein. The two polypeptides may bedirectly linked via a peptide bond or may be separated by a linker,spacer, or a section of a third protein into which they are inserted inframe. In the Examples, an amino acid spacer was used between thepolypeptides. A spacer may be between 2 and 20 amino acids, suitablybetween 4 and 10 amino acids, suitably between 6 and 8 amino acids.Suitably the amino acids in the spacer have a small side chain and arenot charged, such as glycine, alanine or serine. In the Examples, aspacer including two glycine residues, two serine residues and arginineand two more serine residues was used. Those of skill in the art willappreciate other spacers could be used.

In the Examples, the vaccine vectors have the antigenic polypeptides(MPP and/or TRAP polypeptides) and the immunostimulatory polypeptide(either CD154 or HMGB1 or both) encoded on the same polynucleotide andin frame with each other. In alternative embodiments, theimmunostimulatory polypeptide and the antigenic polypeptide may beencoded by distinct polynucleotides. Those of skill in the art willappreciate that a variety of methods may be used to obtain expression ofthe antigenic polypeptide and the HMGB1 polypeptide on the surface ofthe vaccine vector. Such methods are known to those skilled in the art.

Compositions comprising the vaccine vector and a pharmaceuticallyacceptable carrier are also provided. A pharmaceutically acceptablecarrier is any carrier suitable for in vivo administration. Suitably,the pharmaceutically acceptable carrier is acceptable for oral, nasal ormucosal delivery. The pharmaceutically acceptable carrier may includewater, buffered solutions, glucose solutions or bacterial culturefluids. Additional components of the compositions may suitably includeexcipients such as stabilizers, preservatives, diluents, emulsifiers andlubricants. Examples of pharmaceutically acceptable carriers or diluentsinclude stabilizers such as carbohydrates (e.g., sorbitol, mannitol,starch, sucrose, glucose, dextran), proteins such as albumin or casein,protein-containing agents such as bovine serum or skimmed milk andbuffers (e.g., phosphate buffer). Especially when such stabilizers areadded to the compositions, the composition is suitable for freeze-dryingor spray-drying. The vaccine vector in the compositions may not becapable of replication, suitably the vaccine vector is inactivated orkilled prior to addition to the composition.

Methods of enhancing immune responses in a subject by administering avaccine vector are also provided. The vaccine vector may contain a firstpolynucleotide encoding an Aplicomplexan Rhomboid polypeptide and asecond polynucleotide encoding an immunostimulatory polypeptide. Theimmunostimulatory polypeptide is suitably as polypeptide nativelyassociated with a vertebrate immune system and involved in stimulatingan immune response. The immunostimulatory polypeptide may stimulate thenative or adaptive immune response of the subject. Suitably a HMGB1polypeptide or a CD154 polypeptide as described more fully above may beused as the immunostimulatory polypeptide. In the methods providedherein, the vaccine vector comprising an Apicomplexan Rhomboidpolypeptide and an immunostimulatory polypeptide is administered to assubject in an amount effective to enhance or effect an immune responseof the subject to the vaccine vector and in particular to the antigenicRhomboid polypeptide and suitably to the apicomplexan parasite. Theenhanced immune response may include the antibody or T cell response.Suitably the immune response is a protective immune response, but theimmune response may not be fully protective, but may be capable ofreducing the morbidity or mortality associated with infection. Theimmunostimulatory polypeptides may be used to enhance the immuneresponse in the subject to any foreign antigen or antigenic polypeptidepresent in the vaccine vector in addition to the Rhomboid polypeptide.One of skill in the art will appreciate that the immunostimulatorypolypeptide could be used to enhance the immune response to more thanone antigenic polypeptide present in a vaccine vector. Enhancing animmune response includes, but is not limited to, inducing a therapeuticor prophylactic effect that is mediated by the immune system of thesubject. Specifically, enhancing an immune response may include, but isnot limited to, enhanced production of antibodies, enhanced classswitching of antibody heavy chains, maturation of antigen presentingcells, stimulation of helper T cells, stimulation of cytolytic T cellsor induction of T and B cell memory.

Suitably, the vaccine vector contains a polynucleotide encoding apolypeptide including amino acids 150-183 and 89-109 of the HMGB1polypeptide (SEQ ID NO: 15) or a homolog thereof. In the Examples, a 190amino acid polypeptide of HMGB1 was used. Suitably, the polynucleotideencodes a HMGB1 polypeptide from the same species as the subject.Heterologous combinations of HMGB1 polypeptides and subjects (e.g. ahuman HMGB1 polypeptide for use in a chicken vaccine) may be useful inthe methods of the invention because HMGB1 is highly conserved through awide number of species. The HMGB1 polypeptide may be used to enhance theimmune response to more than one antigenic polypeptide present in avaccine vector. The polypeptide from HMGB1 stimulates an immune responseat least in part by activating dendritic cells and macrophages and thusstimulating production of cytokines such as IL-1, IL-6, IFN-γ and TNF-α.In the Examples, a polypeptide of HMGB1 was expressed on the surface ofthe vaccine vector.

The vaccine vector may suitably contain a CD154 polypeptide capable ofbinding to CD40 and activating CD40. The vaccine comprising thepolynucleotide encoding a CD154 polypeptide capable of binding to CD40is administered to a subject in an amount effective to enhance or affectthe immune response of the subject to the vaccine. Suitably, the vaccinecontains a polynucleotide encoding a polypeptide including amino acids140-149 of the human CD154 polypeptide (SEQ ID NO: 25) or a homologthereof. As noted above, a homologue of amino acid 140-149 derived fromone species may be used to stimulate an immune response in a distinctspecies. Suitably, the polynucleotide encodes a CD54 polypeptide fromthe same species as the subject. Suitably, a polynucleotide encoding thepolypeptide of SEQ ID NO: 26 is used in human subjects, a polynucleotideencoding the polypeptide of SEQ ID NO: 27 is used in chickens, apolynucleotide encoding the polypeptide of SEQ ID NO: 28 is used inducks, a polynucleotide encoding the polypeptide of SEQ ID NO: 29 isused in mice, and a polynucleotide encoding the polypeptide of SEQ IDNO: 30 is used in cows. The human CD154 polypeptide (SEQ ID NO: 26) hasbeen used in a chicken vaccine and was demonstrated to enhance theimmune response to a foreign antigen. Thus other heterologouscombinations of CD154 polypeptides and subjects may be useful in themethods of the invention.

In addition, methods of enhancing an immune response against anapicomplexan parasite and methods of reducing morbidity associated withsubsequent infection with an apicomplexan parasite are disclosed.Briefly, the methods comprise administering to a subject an effectiveamount of a vaccine vector comprising a first polynucleotide sequenceencoding an Apicomplexan Rhomboid polypeptide. The vaccine vector mayalso include a second polynucleotide encoding an immunostimulatorypolypeptide in an effective amount. The Rhomboid polypeptides mayinclude SEQ ID NO: 1-4, 37, 38 or combinations or fragments thereof. Theinsertion of the Rhomboid polypeptides into the vector may beaccomplished in a variety of ways known to those of skill in the art,including but not limited to the scarless site-directed mutation systemdescribed in BMC Biotechnol. 2007 Sep. 17: 7(1): 59, Scarless andSite-directed Mutagenesis in Salmonella Enteritidis chromosome, which isincorporated herein by reference in its entirety and the method usedherein as described in Nguyen and Schumann J Biotechnol 2006 122:473-482, which is incorporated herein by reference in its entirety. Thevector may also be engineered to express the Rhomboid polypeptides inconjunction with other antigenic polypeptides from apicomplexanparasites such as TRAP or from other pathogens including viruses such asInfluenza M2e or bacteria such as Salmonella or E. coli. In particular,a polypeptide of CD154 capable of binding CD40 or HMGB1 may be expressedby the vector to enhance the immune response of the subject to theRhomboid polypeptide.

The compositions containing antigenic polypeptides may also be used todecrease the morbidity associated with subsequent infection by anapicomplexan parasite. The compositions may prevent the parasite fromcausing disease or may limit or reduce any associated morbidity in asubject to which the compositions or vaccine vectors described hereinwere administered. The compositions and vaccine vectors described hereinmay reduce the severity of subsequent disease by decreasing the lengthof disease, weight loss, severity of symptoms of the disease, decreasingthe morbidity or mortality associated with the disease or reducing thelikelihood of contracting the disease. The compositions may also reducethe spread of the parasite by inhibiting transmission. The morbidity ormortality associated with the disease after administration of thevaccine vectors described herein may be reduced by 25%, 30%, 40%, 50%,60%, 70%, 80%, 90% or even 100% as compared to similar subjects notprovided the vaccine vector.

For administration to animals or humans, the compositions may beadministered by a variety of means including, but not limited tointranasally, mucosally, by spraying, intradermally, parenterally,subcutaneously, intraperitonelly, intravenously, intracrannially,orally, by aerosol or intraamuscularly. Eye-drop administration, oralgavage or addition to drinking water or food is additionally suitable.For poultry, the compositions may be administered in ovo.

Some embodiments of the invention provide methods of enhancing immuneresponses in a subject. Suitable subjects may include, but are notlimited to, vertebrates, suitably mammals, suitably a human, and birds,suitably poultry such as chickens or turkeys. Other animals such ascows, cats, dogs or pigs may also be used. Suitably, the subject isnon-human and may be an agricultural animal.

The useful dosage of the vaccine to be administered will vary dependingon the age, weight and species of the subject, the mode and route ofadministration and the type of pathogen against which an immune responseis sought. The composition may be administered in an close sufficient toevoke an immune response. It is envisioned that doses ranging from 10³to 10¹⁰ vector copies (i.e. colony forming units or plaque formingunits), from 10⁴ to 10⁹ vector copies, or from 10⁵ to 10⁷ vector copiesare suitable.

The composition may be administered only once or may be administered twoor more times to increase the immune response. For example, thecomposition may be administered two or more times separated by one week,two weeks, three weeks, 1 month, 2 months, months, 6 months, 1 year ormore. The vaccine vector may comprise viable microorganisms prior toadministration, but in some embodiments the vector may be killed priorto administration. In some embodiments, the vector may be able toreplicate in the subject, while in other embodiments the vector may notbe capable of replicating in the subject. Methods of inactivatingmicroorganisms used as vectors are known to those of skill in the art.For example a bacterial vaccine vector may be inactivated usingformalin, ethanol, heat exposure, or antibiotics. Those of skill in theart may use other methods as well.

It is envisioned that several epitopes or antigens from the same ordifferent pathogens may be administered in combination in a singlevaccine to generate an enhanced immune response against multipleantigens. Recombinant vaccines may encode antigens from multiplepathogenic microorganisms, viruses or tumor associated antigens.Administration of vaccine capable of expressing multiple antigens hasthe advantage of inducing immunity against two or more diseases at thesame time. For example, live attenuated bacteria provide a suitablevector for eliciting an immune response against multiple antigens from asingle pathogen, e.g., TRAP (SEQ ID NO: 6) and MPP from Eimeria (SEQ IDNO: 2); or against multiple antigens from different pathogens, e.g.,Eimeria and Influenza or Salmonella.

Vaccine vectors may be constructed using exogenous polynucleotidesencoding antigens which may be inserted into the vaccine vector at anynon-essential site or alternatively may be carried on a plasmid or otherextra chromosomal vehicle (e.g. a BAC or YAC) using methods well knownin the art. One suitable site for insertion of polynucleotides is withinexternal portions of transmembrane proteins or coupled to sequences thattarget the exogenous polynucleotide for secretory pathways and/or allowattachment to the cell wall. One example of a suitable transmembraneprotein for insertion of polynucleotides is the lamB gene. One suitablemethod of cell wall attachment is provided in the Examples

Exogenous polynucleotides include, but are not limited to,polynucleotides encoding antigens selected from pathogenicmicroorganisms or viruses and include polynucleotides that are expressedin such a way that an effective immune response is generated. Suchpolynucleotides may be derived from pathogenic viruses such as influenza(e.g., M2e, hemagglutinin, or neuraminidase), herpesviruses (e.g., thegenes encoding the structural proteins of herpesviruses), retroviruses(e.g., the gp160 envelope protein), adenoviruses, paramyxoviruses,coronaviruses and the like. Exogenous polynucleotides can also beobtained from pathogenic bacteria, e.g., genes encoding bacterialproteins such as toxins, outer membrane proteins or other highlyconserved proteins. Further, exogenous polynucleotides from parasites,such as other Apicomplexan parasites are attractive candidates for usein a vector vaccine.

The present disclosure is not limited to the specific details ofconstruction, arrangement of components, or method steps set forthherein. The compositions and methods disclosed herein are capable ofbeing made, practiced, used, carried out and/or formed in various waysthat will be apparent to one of skill in the art in light of thedisclosure that follows. The phraseology and terminology used herein isfor the purpose of description only and should not be regarded aslimiting to the scope of the claims. Ordinal indicators, such as first,second, and third, as used in the description and the claims to refer tovarious structures or method steps, are not meant to be construed toindicate any specific structures or steps, or any particular order orconfiguration to such structures or steps. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, istended merely to facilitate the disclosure and does not imply anylimitation on the scope of the disclosure unless otherwise claimed. Nolanguage in the specification, and no structures shown in the drawings,should be construed as indicating that any non-claimed element isessential to the practice of the disclosed subject matter. The useherein of the terms “including,” “comprising,” or “having,” andvariations thereof, is meant to encompass the elements listed thereafterand equivalents thereof, as well as additional elements. Embodimentsrecited as “including,” “comprising,” or “having” certain elements arealso contemplated as “consisting essentially of” and “consisting of”those certain elements. The terms “a” “an” and “the” may mean one ormore than one unless specifically delineated.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. For example, if a concentration range isstated as 1% to 50%, it is intended that values such as 2% to 40%, 10%to 30%, or 1% to 3%, etc., are expressly enumerated in thisspecification. These are only examples of what is specifically intended,and all possible combinations of numerical values between and includingthe lowest value and the highest value enumerated are to be consideredto be expressly stated in this disclosure. Use of the word “about” todescribe a particular recited amount or range of amounts is meant toindicate that values very near to the recited amount are included inthat amount, such as values that could or naturally would be accountedfor due to manufacturing tolerances, instrument and human error informing measurements, and the like. All percentages referring to amountsare by weight unless indicated otherwise.

The following examples are meant only to be illustrative and are notmeant as limitations on the scope of the invention or of the appendedclaims. All references, included patents, patent publications andnon-patent literature, cited herein are hereby incorporated by referencein their entirety. Any conflict between statements in references andthose made herein should be resolved in favor of the statementscontained herein.

EXAMPLES Example 1 Construction of Vaccine Vectors

Multiple combinations of vaccine were constructed for the purpose oftesting efficacy and determining the influence of each on protectionagainst Eimeria maxima challenge. A cartoon showing the constructs usedin the examples is shown as FIG. 2. The TRAP MPP HMGB1, and MPP HMGB1sequences were synthesized and inserted into pNDH10 plasmid for cellsurface expression. Each sequence was synthesized with a BamHIrestriction site the 5′ end and an AatII restriction site at the 3 endimmediately adjacent to the fibronectin binding protein B (fbpB).Expression of the vaccine sequence and fbpB is regulated by a sortingmotif that operon previously inserted into pNDH10 plasmid [1]. The fbpBincluded a sorting motif that was recognized by sortase A that anchorsthe fbpB to the cell surface of a sortase A expressing bacterium [1].Thus, the vaccine sequences are placed upstream and in frame with thefbpB sequence such that when the fbpB is anchored to sortase A on thecell wall the vaccine vector sequence will be expressed on the surfaceof the bacteria. Plasmid pNDH10 containing the vaccine sequence, fbpB,and xyl operon was transformed into Bacillus subtilis 1A857 expressingsortase [2]. Each plasmid was transformed into 1A857 by adding 0.6 μginsert/plasmid into a competent 1A857 culture with 0.1 M ethylene glycoltetraacetic acid (EGTA). After transformation, 1A857 expressing pNDH10were selected on LB agar containing 5 μg/mL chloramphenicol to selectonly cells that carried antibiotic resistance conferred by the plasmidvia a cat sequence that encodes chloramphenicol acetyl transferase.Bacillus subtilis 1A857 transformed with MPP HMGB1 (SEQ ID NO: 33), orTRAP MPP HMGB1 (SEQ ID NO: 31) pNDH10 plasmids were confirmed by plasmidextraction followed by PCR. Each 1A857/pNDH10/insert construct was grownand induced in 0.6% xylose in LB broth +0.1% glucose with 5 μg/mLchloramphenicol for 9 h at 37° C. while shaking, MPP-HMGB1 (SEQ ID NO:34) and TRAP-MPP-HMGB1 (SEQ ID NO: 32) protein expression were confirmedby Western blot and indirect fluorescence microscopy with rabbitanti-HMGB1 antibodies.

Example 2 Reduced Morbidity and Mortality of Chicks after EimeriaInfection

Vectored vaccines MPP HMGB1 and TRAP MPP HMGB1 were tested for abilityto provide protection against an Eimeria maxima challenge whenadministered through the drinking water in conjunction with a modifiedchitosan adjuvant. Broiler chicks were vaccinated at 4 and 14 days ofage with the respective vaccine in the drinking water at a dilution of1:128 (5×10⁵ cfu/chick) for 24 h. At 21 d of age, all groups wereweighed and challenged with 4×10⁴ sporulated oocysts of E. maxima byoral gavage. At 28 d of age, body weight (BW) and body weight gain ofsurvivors (BWG) were recorded during the challenge period. Additionally,mortality was documented to determine vaccine candidate efficacy. Eightdays post-challenge BW was significantly higher in chicks vaccinatedwith TRAP-MPP-HMGB1 and MPP-HMGB1 when compared with non-vaccinatedchicks (FIG. 3). BWG was significantly higher for ail vaccinated groups8 d post-challenge when compared to controls (FIG. 4). Mortality wasalso significantly lower in the TRAP-MPP-HMGB1 and MPP-HMGB1 vaccinatedgroups with the unvaccinated group (FIG. 5).

-   [1] Kim L, Mogk A Schumann W. A xylose-inducible Bacillus subtilis    integration vector and its application. Gene 1996 Nov. 28;    181(1-2):71-6.-   [2] Nguyen H D, Schumann W. Establishment of an experimental system    allowing immobilization of proteins on the surface of Bacillus    subtilis cells. Journal of biotechnology 2006 Apr. 20; 122(4):    473-82.

We claim:
 1. A vaccine vector comprising a first polynucleotide sequenceencoding an Apicomplexan Rhomboid polypeptide expressed on the surfaceof the vaccine vector, wherein the Rhomboid polypeptide consists of apolypeptide having greater than 90% sequence identity to SEQ ID NO: 2 oran immunogenic fragment of SEQ ID NO: 2 comprising amino acids 1-11,18-27, or 31-43 of SEQ ID NO: 2, and wherein the vaccine vectorcomprises a bacterial, yeast, viral or liposome-based vector.
 2. Thevaccine vector of claim 1, further comprising a second polynucleotidesequence encoding an immunostimulatory polypeptide, wherein theimmunostimulatory polypeptide is expressed on the surface of the vaccinevector, and wherein an immunostimulatory polypeptide comprises apolypeptide capable of stimulating a naïve or adaptive immune response.3. The vaccine vector of claim 2, wherein the immunostimulatorypolypeptide comprises an HMGB1 polypeptide.
 4. The vaccine vector ofclaim 3, wherein the HMGB1 polypeptide comprises a polypeptide selectedfrom the group consisting of SEQ ID NOs: 15-23, a polypeptide having atleast 95% sequence identity to SEQ ID NO: 15-23 and combinationsthereof.
 5. The vaccine vector of claim 2, wherein the immunostimulatorypolypeptide comprises a CD154 polypeptide capable of binding CD40, theCD154 polypeptide having fewer than 50 amino acids and comprising aminoacids 140-149 of a polypeptide selected from the group consisting of SEQID NO: 24, SEQ ID NO: 25, or is a polypeptide selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30 and polypeptides having at least 90% sequence identityto at least one of SEQ ID NOs: 26-30.
 6. The vaccine vector of claim 2,wherein the vector comprises more than one copy of the firstpolynucleotide or more than one copy of the second polynucleotidesequence.
 7. The vaccine vector of claim 2, wherein the firstpolynucleotide sequence is linked in the same reading frame to thesecond polynucleotide sequence.
 8. The vaccine vector of claim 7,wherein the first polynucleotide and the second polynucleotide arelinked via a spacer nucleotide sequence.
 9. The vaccine vector of claim1, wherein the vaccine vector is selected from the group consisting of avirus, a bacterium, a yeast and a liposome.
 10. The vaccine vector ofclaim 9, wherein the vaccine vector is a Bacillus spp.
 11. The vaccinevector of claim 1, further comprising a third polynucleotide encoding aTRAP polypeptide selected from the group consisting of polypeptideshaving at least 95% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7, and SEQ ID NO:
 40. 12. The vaccine vector of claim 2, whereinthe first polynucleotide and the second polynucleotide encode apolypeptide selected from the group consisting of SEQ ID NO: 32, SEQ IDNO: 34 and a polypeptide having 95% sequence identity to SEQ ID NO: 32or SEQ ID NO:
 34. 13. A pharmaceutical composition comprising thevaccine vector of claim 1 and a pharmaceutically acceptable carrier.